ABSTRACT Reactive oxygen species are induced by both endogenous and exogenous pathways and they can induce covalent modifications to DNA. The long-term objective of this application is to understand the biological implications of DNA damage induced by reactive oxygen species, and we have made significant progresses in the last funding cycle. In this renewal, we propose to determine the structures of new oxidative intrastrand crosslink lesions by NMR and mass spectrometry. We will also employ LC-MS/MS with the isotope-dilution method to quantify intrastrand crosslink lesions formed from [unreadable] radiation, Fenton reaction, and oxidants produced by activated human phagocytic cells. In addition, we are going to construct oligodeoxyribonucleotides harboring site-specifically incorporated oxidative DNA lesions and use them as substrates for in-vitro replication studies. We will also insert these oxidative lesions DNA into single- and double-stranded shuttle vectors, allow them to propagate in bacterial and human cells, and identify and quantify the replication products by employing a newly developed mass spectrometric assay. Furthermore, we are going to examine how 6-thio-2'-deoxyguanosine and its oxidation/methylation products perturb the methylation of cytosine residues at CpG site and affect the binding of DNA to methyl-CpG binding proteins. The outcome of the proposed research will provide significant new knowledge about the biological consequences of nucleic acid damage induced by reactive oxygen species and it may also lead to the discovery of novel mechanisms for the cytotoxic effects of 6-thioguanine and its analogs, which are commonly prescribed anticancer and immunosuppressive drugs.